Abstract

The neuromodulator acetylcholine (ACh) is crucial for several cognitive functions, such as perception, attention, and learning and memory. Whereas, in most cases, the cellular circuits or the specific neurons via which ACh exerts its cognitive effects remain unknown, it is known that auditory cortex (AC) neurons projecting from layer 5B (L5B) to the inferior colliculus, corticocollicular neurons, are required for cholinergic-mediated relearning of sound localization after occlusion of one ear. Therefore, elucidation of the effects of ACh on the excitability of corticocollicular neurons will bridge the cell-specific and cognitive properties of ACh. Because AC L5B contains another class of neurons that project to the contralateral cortex, corticocallosal neurons, to identify the cell-specific mechanisms that enable corticocollicular neurons to participate in sound localization relearning, we investigated the effects of ACh release on both L5B corticocallosal and corticocollicular neurons. Using in vitro electrophysiology and optogenetics in mouse brain slices, we found that ACh generated nicotinic ACh receptor (nAChR)-mediated depolarizing potentials and muscarinic ACh receptor (mAChR)-mediated hyperpolarizing potentials in AC L5B corticocallosal neurons. In corticocollicular neurons, ACh release also generated nAChR-mediated depolarizing potentials. However, in contrast to the mAChR-mediated hyperpolarizing potentials in corticocallosal neurons, ACh generated prolonged mAChR-mediated depolarizing potentials in corticocollicular neurons. These prolonged depolarizing potentials generated persistent firing in corticocollicular neurons, whereas corticocallosal neurons lacking mAChR-mediated depolarizing potentials did not show persistent firing. We propose that ACh-mediated persistent firing in corticocollicular neurons may represent a critical mechanism required for learning-induced plasticity in AC.

SIGNIFICANCE STATEMENT Acetylcholine (ACh) is crucial for cognitive functions. Whereas in most cases the cellular circuits or the specific neurons via which ACh exerts its cognitive effects remain unknown, it is known that auditory cortex (AC) corticocollicular neurons projecting from layer 5B to the inferior colliculus are required for cholinergic-mediated relearning of sound localization after occlusion of one ear. Therefore, elucidation of the effects of ACh on the excitability of corticocollicular neurons will bridge the cell-specific and cognitive properties of ACh. Our results suggest that cell-specific ACh-mediated persistent firing in corticocollicular neurons may represent a critical mechanism required for learning-induced plasticity in AC. Moreover, our results provide synaptic mechanisms via which ACh may mediate its effects on AC receptive fields.

Here, we investigated the effects of exogenous and endogenous ACh on the synaptic excitability of L5B corticocollicular neurons. However, L5B contains additional types of projection neurons, including corticocallosal neurons, a second major class of L5 neurons with axons projecting to the contralateral A1. Because recent studies in a variety of cortical areas, including the AC, revealed numerous differences in the physiological properties of pyramidal tract (PT) neurons, of which corticocollicular are a subtype, and intratelencephalic (IT) neurons, of which corticocallosal neurons are a subtype (for review, see Shepherd, 2013), we hypothesized that ACh may have cell-specific effects on the excitability of projection neurons. These cell-specific effects may be important for the cholinergic-mediated experience-dependent plasticity involved in relearning sound localization after plugging one ear (Bajo et al., 2010), as well as in the distinct role of PT and IT neurons in the delay period that occurs during movement (Li et al., 2015).

To study the effects of ACh on corticocollicular and L5B corticocallosal neurons, we used in vivo retrograde labeling, as well as in vitro electrophysiological methods combined with optogenetic activation of cholinergic fibers. We show that ACh has cell-specific effects on L5B projection neurons. Namely, ACh elicits nicotinic ACh receptor (nAChR)-mediated depolarizing potentials in both neuronal types, whereas ACh evokes muscarinic ACh receptor (mAChR)-mediated hyperpolarizing potentials in corticocallosal neurons, but long-lasting mAChR-mediated depolarizing potentials only in corticocollicular neurons. The long-lasting mAChR-mediated depolarizing potential generates persistent firing in corticocollicular neurons, which may be involved in top-down modulation of auditory learning.

Materials and Methods

Animals.

ICR mice (Harlan Laboratories) and Chat-ChR2-EYFP mice (The Jackson Laboratory) of either sex at age P22–P40 for microsphere injection and P24–P45 for recordings were used for experiments that examined the effect of endogenous release of ACh on corticocollicular and L5B corticocallosal neurons. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh.

Stereotaxic injections.

Mice were anesthetized with isofluorane (induction: 3% in O2, 0.6 L/min; maintenance: 50% of induction dose) and positioned in a stereotaxic frame (Kopf Instruments). Projection neurons in the AC were labeled retrogradely by injecting different colored fluorescent latex microspheres (Lumafluor) in the contralateral AC (in a small craniotomy drilled 4 mm posterior to bregma and 4 mm lateral, injection depth 1 mm) and the ipsilateral IC (1 mm posterior to lambda and 1 mm lateral, injection depth 0.75 mm). A volume of ∼ 0.1 μl microspheres was pressure injected (25 psi, 10–15 ms duration) from capillary pipettes (Drummond Scientific) with a Picospritzer (Parker–Hannifin). The injection volume was distributed between several sites along the injection depth so as to label the entire extent of the injection site. After injection, the pipette was held in the brain for 1.5 min before slowly withdrawing. The animals were allowed to recover for at least 48 h to allow time for retrograde transport of the tracers.

To test for persistent firing, neurons were depolarized by a current injection to a membrane potential that was close to firing threshold. Firing frequency of the action potentials (APs) elicited in response to ACh release was plotted as a function of time starting from the stimulus onset (time, t = 0) to 15 s after the termination of the stimulus. Persistent firing was defined as the ability of the neuron to fire APs at least 5 s after the stimulus (t = 0) termination. AP threshold was measured in phase plane as the membrane potential at which the depolarization slope shows the first abrupt change (Δslope > 10 V/s). AP width was calculated as the full-width at the half-maximum amplitude of the AP (peak minus threshold). Input resistance, Ri, was calculated in voltage- or current-clamp mode by giving a −5 mV or −5 pA step, which resulted in transient current or voltage responses. In voltage-clamp mode, the difference between baseline and steady-state hyperpolarized current (ΔI) was used to calculate Ri using the following formula: Ri = −5 mV/ΔI − Rseries. In current-clamp mode, the difference in the steady-state voltage and baseline voltage (ΔV) was used to calculate Ri using the following formula: Ri = ΔV/−5 pA. The average Vm was calculated by holding the neuron in voltage-follower mode (current clamp at I = 0) immediately after breaking in and averaging the membrane potential over the next 20 s.

Pharmacology.

The identity of the receptors mediating the responses elicited by the release of ACh on corticocollicular or L5B corticocallosal neurons was established by applying blockers of nAChRs and mAChRs (nAChR-mediated responses identified by the application of a mixture of nAChR blockers mecamylamine hydrochloride (5 μm) + hexamethonium bromide (50 μm), or dihydro-β-erythroidine (DHβE) (500 nm); mAChR-mediated responses identified by the application of atropine (1 μm). The blockers were applied for at least 10 min before assessing their effects on the evoked responses. All drugs were obtained from Sigma-Aldrich.

Puffing experiments.

After establishing whole-cell recording from corticocallosal or corticocollicular neurons, 100 μm ACh was puffed for 20 ms at 20 psi from a patch pipette placed 50 μm from the neuronal soma. The 100 μm ACh concentration was used because high concentrations of agonists (>100 μm ACh) affect nAChR-mediated responses by either desensitizing the receptors or causing an open channel block (Quick and Lester, 2002). Responses were identified if they were >2.5 SDs of the baseline noise level (noise levels were measured during a 100 ms timing period before the ACh puff) and were further analyzed.

Optogenetic simulation.

After establishing whole-cell recordings from corticocallosal or corticocollicular neurons, we used wide-field illumination (using a 40× objective) with a blue LED (470 nm at maximum intensity; Thor Laboratories) to activate ChR2-containing cholinergic axons. To assess the effects of endogenous release of ACh on A1 L5B corticocallosal neurons, stimulations ranged from a single 5 ms pulse of blue light to 10 or 60 pulses (5 ms each, 50 Hz). Responses were identified if they were >2.5 SDs of the baseline noise level (noise levels were measured during a 100 ms timing period before illumination) and were further analyzed.

Anatomy.

For anatomical visualization of EYFP-containing cholinergic axons among corticocollicular and L5B corticocallosal neurons, ChAT-ChR2-EYFP mice were injected with red fluorescent latex microspheres in the IC to label corticocollicular neurons and cholera toxin subunit B (CTB, far red emission) in the contralateral AC to label corticocallosal neurons. Mice were allowed to recover for 7 d and were subsequently perfused with 4% paraformaldehyde (PFA). The brains were extracted and postfixed in 4% PFA for 4 h, after which they were cryoprotected overnight in 25% sucrose solution maintained at 4°C. The brains were washed with phosphate buffer solution the next day and were sectioned on a microtome into 50-μm-thick sections containing the AC. The sections were mounted and imaged on an Olympus microscope with a 20× objective using standard filters for green, red, and far-red emissions. The acquired images were subsequently processed in ImageJ for brightness and contrast.

Statistical analysis.

Student's t tests were used for statistical comparisons between different population of neurons. Paired t tests were used for all statistical analyses to compare the effect of drug applications on responses generated by exogenous or endogenous release of ACh. In cases in which two drugs were added sequentially, a one-way ANOVA was used to examine the effect of each drug application on the response. Significance was reported if the p-value was found to be <0.05.

Next, we determined whether endogenous release of ACh on AC L5B corticocallosal and corticocollicular neurons has actions similar to those of exogenous ACh. To investigate the effect of endogenous ACh, we used the ChAT-ChR2-EYFP mouse line, which expresses the light-activated cation channel channelrhodopsin (ChR2) selectively in cholinergic axons (Zhao et al., 2011). The ChAT-ChR2-EYFP mouse line carries several copies of the vesicular ACh transporter gene (VAChT), which leads to overexpression of functional VAChT and to a threefold increase in ACh release in these mice compared with control mice (Kolisnyk et al., 2013). Although this VAChT overexpression may contribute to cellular outputs that differ from the outputs due to normal levels of ACh release, we used this mouse cell line because it can reveal cell-specific effects of endogenous ACh release between L5B corticocollicular and corticocallosal neurons and further test the validity of the differential effect of exogenous ACh application on corticocollicular and L5B corticocallosal excitability.

To confirm the presence of ChR2-EYFP fibers among AC L5B corticocallosal and corticocollicular neurons, we performed in vivo injections of fluorescent retrograde tracers in ChAT-ChR2-EYFP mice. Small volumes of red fluorescent microspheres (red emission) were injected in the IC, whereas CTB (far red emission) was injected in the contralateral AC (Fig. 3A). Mice were perfused 1 week later and then their brains were cryoprotected and subsequently sectioned into 50-μm-thick AC-containing sections. Epifluorescence imaging revealed labeled corticocollicular neurons in L5B of the AC (Fig. 3B), whereas CTB-labeled corticocallosal neurons were present in L5B and other layers of the AC (Fig. 3C). EYFP-containing green cholinergic axons were also present in L5B AC (Fig. 3D). An overlay of the three separate images (Fig. 3B–D) revealed an intermingled population of AC L5B corticocollicular and corticocallosal neurons (red and blue) among green cholinergic axons (Fig. 3E), thus confirming the presence of cholinergic axons expressing ChR2 in AC L5B.

ChR2-EYFP fibers among AC L5B corticocallosal and corticocollicular neurons in the ChAT-ChR2-EYFP mouse line. A, Labeling of corticocollicular and corticocallosal neurons with fluorescent tracers. Projection neurons in the AC were labeled by injecting different colored retrograde tracers in the contralateral AC (choleratoxin to label corticocallosal neurons) and the ipsilateral IC (red fluorescent microspheres to label corticocollicular neurons). B, A 20× epifluorescence image showing labeled corticocollicular neurons in L5B of the AC. C, A 20× epifluorescence image showing labeled corticocallosal neurons in L5B and other layers of the AC. D, A 20× epifluorescence image showing green cholinergic axons in L5B of AC. E. Merged image (B–D combined) showing intermingled population of corticocallosal and corticocollicular neurons among green cholinergic axons in L5B of AC.

To assess the effects of endogenous release of ACh on AC L5B corticocallosal neurons, we used wide-field illumination of the slice with a blue LED (λ = 470 nm) to activate ChR2-containing cholinergic terminals and evoke ACh release. In 12 of 21 corticocallosal neurons, endogenous release of ACh by stimulation with a single pulse of blue light (pulse width = 5 ms) generated a monophasic depolarizing potential (Fig. 4A1, control black trace), which was similar to the monophasic depolarizing potential that we observed with exogenous ACh. To assess the pharmacology of these responses, we stimulated with 10 pulses of blue light (at 50 Hz) because the responses were more robust. This monophasic depolarizing potential was mediated by nAChRs because it was blocked by the application of nAChR blockers (Fig. 4A2, orange trace, A4). Furthermore, the depolarizing potential was also blocked by 500 nm DHβE, indicating that it was mediated by α4β2 nAChRs (Fig. 4A3, magenta trace, A4).

In four of 21 corticocallosal neurons, endogenous release of ACh with a single pulse of blue light generated biphasic responses: a depolarizing potential followed by a hyperpolarizing potential (Fig. 4B1, control black trace). Sequential application of 1 μm atropine and nAChR blockers abolished the hyperpolarizing and depolarizing potential, respectively, showing that the hyperpolarizing phase is mediated mAChRs and the depolarizing phase is mediated by nAChRs (Fig. 4B2, control black trace = depolarization/hyperpolarization, green trace = after atropine, orange trace = after nAChR blockers; B3, summary of the effect of atropine on the hyperpolarizing potential; B4, summary of the effect of atropine and nAChR blockers on the depolarizing potential). Finally, in five of 21 corticocallosal neurons, endogenous release of ACh by optogenetic stimulation with a single pulse of blue light generated a hyperpolarizing potential (Fig. 4C1, control black trace). This hyperpolarizing potential was mediated by mAChRs because it was abolished by the application of 1 μm atropine (Fig. 4C2, control black trace = hyperpolarizing potential, green trace = atropine, C3). Although we did not observe monophasic hyperpolarizing potentials after extracellular ACh application, together, our results indicate that exogenous and endogenous ACh had similar effects on the excitability of L5B corticocallosal neurons.

Next, we studied the effect of endogenous ACh on the excitability of corticocollicular neurons. In five of 13 corticocollicular neurons, one or 10 pulses of blue light elicited monophasic depolarizing responses (Fig. 5A1). Because corticocollicular neurons consistently gave responses to 60 pulses delivered at 50 Hz, we used this stimulation protocol for assessing the response of corticocollicular neurons to endogenous ACh. Under these conditions, in seven of 13 corticocollicular neurons, endogenous release of ACh generated a monophasic depolarizing potential, which was similar to the monophasic depolarizing potential that we observed with exogenous ACh (Fig. 5A2, control black trace). This depolarizing potential was mediated by α4β2 nAChRs because it was blocked by DHβE (Fig. 5A2, magenta trace, A3). In one of five corticocollicular neurons that showed a depolarizing potential, application of nAChR blockers eliminated the depolarizing potential and revealed a hyperpolarizing potential, which was blocked by the application of atropine (Fig. 5B), indicating that it was mediated by mAChRs. These results show that the responses due to endogenous ACh resemble the responses obtained with exogenous application of ACh.

No evidence for subdivision of corticocollicular neurons based on the variability of their intrinsic properties

Because approximately half of the recorded corticocollicular neurons displayed long-lasting depolarizing potentials after exogenous or endogenous ACh application, we tested whether these neurons comprised a distinct subgroup within the corticocollicular neuronal population. Because corticocollicular and corticocallosal L5B neurons in mouse AC display distinct dendritic morphology and distinct intrinsic properties such as resting membrane potential, input resistance, AP threshold, and AP width (Joshi et al., 2015), we tested whether differences in these properties are associated with the ability of a subpopulation of corticocollicular neurons to generate long-lasting mAChR-mediated depolarizing potentials. Our results showed that the average input resistance, resting membrane potential, AP width, and AP threshold were not different between the corticocollicular neurons displaying long-lasting depolarization (broad) and the corticocollicular not displaying long-lasting depolarization (narrow; Fig. 6A1–A4). This finding is inconsistent with potential further subdivision of the corticollicular neuronal population. To further validate the lack of subdivision, we tested for potential correlations between the variability of these intrinsic parameters and the amplitude of the narrow and broad ACh-mediated responses. Because all corticocollicular neurons displayed fast onset (rapid) cholinergic depolarization, we tested whether there is any correlation between the amplitude of the early depolarization and the observed variability in intrinsic properties. Aside from an expected correlation between the variability of input resistance and the amplitude (1st amplitude) of the early cholinergic response in the neurons not displaying the long-lasting depolarization (Fig. 6B1, black, p = 0.007), the variability of the intrinsic properties of either group of corticocollicular neurons was not correlated with the amplitude of the early cholinergic depolarization (Fig. 6B1–B4). Furthermore, for the corticocollicular neurons displaying the long-lasting depolarization, we observed no significant correlation between the amplitude of this response (2nd amplitude) and the variability of the intrinsic properties of these neurons (Fig. 6C1–C4). Combined with our previous study showing homogeneity in the dendritic morphology of the population of corticocollicular neurons (Joshi et al., 2015), our results suggest that the variability in the intrinsic properties and morphology of corticocollicular neurons is not correlated with the presence or absence of the long-lasting cholinergic depolarization that we observed.

Consistent with our hypothesis, puffing or endogenous release of ACh onto corticocallosal neurons with monophasic depolarizing potentials or biphasic (depolarizing/hyperpolarizing potentials) responses (Fig. 7A1,B1,C1), when held at subthreshold but close to threshold potential, elicited transient firing, but failed to elicit any persistent firing (Fig. 7A2,B2,C2). Plots of the firing frequency as a function of time indicated that corticocallosal neurons fired APs only during the stimulus or immediately after stimulus termination (Fig. 7A3,B3,C3). Note that even stimulation with 60 pulses did not induce persistent firing in corticocallosal neurons (Fig. 7C4–C6). Similar results were obtained from exogenous application (Fig. 8A1–A3) or endogenous release of ACh (Fig. 8B1–B3) onto corticocollicular neurons exhibiting monophasic depolarizing potentials. However, the subset of corticocollicular neurons exhibiting two-peak depolarizing potentials or broad depolarizing potentials (Fig. 9A1,B1) showed persistent firing in response to either exogenous or endogenous ACh, respectively (Fig. 9A2,B2). Plots of the firing frequency as a function of time indicated that corticocollicular neurons that exhibited two-peak depolarizing potentials or broad depolarizing potentials fired APs for >10 s after the termination of the stimulus (Fig. 9A3,B3). Persistent firing was abolished upon application of atropine, suggesting that mAChRs are crucial for the persistent firing of corticocollicular neurons (Fig. 9A4–A5, B4–B5). Finally, the intrinsic properties, such as AP threshold and AP width, did not change from the onset of firing and during the spike train (Fig. 9C1–C2), suggesting that mAChRs promote persistent firing without affecting the intrinsic AP properties. This finding suggests that ACh is capable of converting AC neurons projecting to the IC into a “persistent activity” mode, whereas intracortically projection neurons do not enter this mode. The persistent firing may be essential for the ACh-dependent, learning-induced plasticity mediated by corticocollicular neurons (Bajo et al., 2010; Leach et al., 2013).

Lack of persistent firing in L5B corticocallosal neurons. A1, Representative example of a corticocallosal neuron held at ∼−70 mV, which responds with a monophasic depolarizing potential to a single puff of 100 μm ACh, denoted by the black arrow. A2, Same neuron as in A1, when held at subthreshold but closer to threshold potential, fires transiently in response to a puff of ACh. This transient spiking was observed in seven of seven corticocallosal neurons with monophasic depolarizing potentials. A3, Average firing frequency quantified for 15 s starting at the time of stimulus onset (t = 0) for APs as in A2 (n = 7). B1, An example of a corticocallosal neuron held at ∼−70 mV, which responds with a biphasic potential to a puff of 100 μm ACh. B2, Same neuron as in B1, when held at subthreshold but closer to threshold potential, fires transiently in response to a puff of ACh. This transient spiking was observed in five of five corticocallosal neurons with biphasic responses. B3, Average firing frequency quantified for 15 s starting at the time of stimulus onset (t = 0) for APs as in B2 (n = 5). C1, Representative example of a corticocallosal neuron held at ∼−70 mV, which responds with a monophasic depolarizing potential in response to optogenetic stimulation with 10 pulses of blue light (λ = 470 nm, pulse width = 5 ms @ 50 Hz). C2, Same neuron as in C1, when held at subthreshold but closer to threshold potential, fires transiently in response to the same optogenetic stimulation used in C1. This transient spiking was observed in four of four corticocallosal neurons with monophasic depolarizing potentials. C3, Average firing frequency quantified for 15 s starting at the time of stimulus onset (t = 0) for APs as in C2 (n = 4). C4, Same corticocallosal neuron as in C1 responds with a broader monophasic depolarizing potential in response to optogenetic stimulation with 60 pulses of blue light (λ = 470 nm, pulse width = 5 ms at 50 Hz). C5, Same neuron as in C4, when held at subthreshold but closer to threshold potential, fires transiently in response to the same optogenetic stimulation used in C4. C6, Average firing frequency quantified for 15 s starting at the time of stimulus onset (t = 0) for APs as in C5 (n = 5).

Lack of persistent firing in L5B corticocollicular neurons exhibiting ACh-evoked monophasic depolarizing potentials. A1, Representative example of a corticocollicular neuron held at ∼−70 mV, which responds with a monophasic depolarizing potential to a puff of 100 μm ACh. A2, Same neuron as in A1, when held at subthreshold but closer to threshold potential, fires transiently in response to a puff of ACh. This transient spiking was observed in five of five corticocollicular neurons exhibiting monophasic depolarizing potentials. A3, Average firing frequency quantified for 15 s starting at the time of stimulus onset (t = 0) for APs as in A2 (n = 5). B1, Representative example of a corticocollicular neuron held at ∼−70 mV, which responds with a monophasic depolarizing potential to the release of endogenous ACh by optogenetic stimulation with 60 pulses of blue light. B2, Same neuron as in B1, when held close to its AP threshold, fires transiently in response to the same optogenetic stimulation used in B1. This transient spiking was observed in three of three corticocollicular neuron with a monophasic depolarizing potentials. B3, Average firing frequency quantified for 15 s starting at the time of stimulus onset (t = 0) for APs as in B2 (n = 3).

Persistent firing in L5B corticocollicular neurons exhibiting ACh-evoked two-peak, broad depolarizing potentials depends on mAChRs. A1, Representative example of a corticocollicular neuron held at ∼−70 mV, which responds with a two-peak depolarizing potential to a puff of ACh. A2, Same neuron as in A1, when held at subthreshold but closer to threshold potential, fires persistently in response to a puff of ACh. This persistent firing was observed in five of five corticocollicular neurons with two-peak depolarizing potentials. A3, Average firing frequency quantified for 15 s starting at the time of stimulus onset (t = 0) for trains of APs as generated in A2 (n = 5). A4, After the application of 1 μm atropine, the same neuron as in A1 and A2 fails to fire persistently in response to a puff of ACh. This effect of atropine was seen in three of three persistently firing corticocollicular neurons with two-peak depolarizing potentials. A5, Average firing frequency quantified for 15 s starting at the time of stimulus onset, before and after the application of atropine (average firing frequency for the last 5 s (t = 10–14) in control: 1.52 ± 0.34; in atropine: 0.00 ± 0.00, n = 3, p < 0.05). B1, Representative example of a corticocollicular neuron held at ∼−70 mV, which responds with a broad depolarizing in response to optogenetic stimulation with 60 pulses of blue light. B2, Same neuron as in B1, when held at subthreshold but closer to threshold potential, fires persistently in response to the same optogenetic stimulation used in B1. This persistent firing was observed in four of four corticocollicular neurons with broad depolarizing potentials. B3, Average firing frequency quantified for 15 s starting at the time of stimulus onset for trains of APs as generated in B2 (n = 4). B4, After the application of 1 μm atropine, the same neuron as in B1 and B2 fails to fire persistently in response to the same optogenetic stimulation used in B1 and B2. This effect of atropine was seen in three of three persistently firing corticocollicular neurons with broad depolarizing potentials. B5, Average firing frequency quantified for 15 s starting at the time of stimulus onset before and after the application of atropine (average firing frequency for the last 5 s in control: 2.36 ± 0.54; in atropine: 0.00 ± 0.00, n = 3, p < 0.05). C1, C2, AP properties during persistent firing. Persistent firing was evoked in response to optogenetic stimulation as in Figure 8B2. Inset shows representative AP waveform. C1, Average of the AP threshold plotted as a function of time during persistent firing (average AP threshold during stimulus (t = 0–2): −40.63 ± 1.55, average AP threshold during the last five seconds of the train (t = 10–14): −39.62 ± 1.36, n = 4, p = 0.16). C2, Average of the AP width plotted as a function of time during persistent firing (average AP width during stimulus (t = 0–2): 1.70 ± 0.12, average AP width during the last 5 s (t = 10–14): 1.77 ± 0.16, n = 4, p = 0.22).

Discussion

To assess the effects of ACh on the excitability of L5B projection neurons, we used in vivo retrogradely fluorescent labeling to label corticocollicular and L5B corticocallosal neurons selectively, single-cell electrophysiology, exogenous application of ACh, and selective stimulation of cholinergic fibers. Whereas exogenous and endogenous ACh generated fast nAChR-mediated depolarizing potentials in corticocollicular and corticocallosal neurons, ACh release generated mAChR-mediated hyperpolarizing potentials in corticocallosal neurons, but long-lasting mAChR-mediated depolarizing potentials in corticocollicular neurons. The long-lasting mAChR-mediated depolarizing potentials were crucial for the persistent firing observed selectively in corticocollicular neurons, which may be involved in auditory learning.

ACh-mediated persistent firing in other brain areas relies either on mAChR-mediated enhancement of postsynaptic Ca2+ and enhancement of afterdepolarizing potentials generated by a Ca2+ -activated nonselective cation current, or voltage-dependent increase in input resistance mediated by a reduction in afterhyperpolarization potentials, or muscarinic mediated inhibition of M-type (KCNQ) potassium channels, or activation of nAChRs and elevations in postsynaptic Ca2+ (McCormick and Prince, 1985; McCormick and Prince, 1986; McCormick and Williamson, 1989; Haj-Dahmane and Andrade, 1999; Egorov et al., 2002; Yamashita and Isa, 2003a, 2003b; Delmas and Brown, 2005; Egorov et al., 2006; Zhang and Séguéla, 2010; Rahman and Berger, 2011; Hedrick and Waters, 2015). Our results show that the persistent firing activity of corticocollicular neurons in response to ACh release is mediated by mAChRs. Whereas our studies did not evaluate the role of postsynaptic Ca2+, because mAChR activation does not affect the spiking properties of corticocollicular neurons and because the time course of persistent firing matches the time course of the mAChR-mediated prolonged depolarization, we propose that it is this depolarization that generates persistent firing lasting for ∼10–20 s after stimulus termination. Our studies did not assess whether this depolarization generates persistent firing in a cell autonomous manner; however, our findings support an additional mechanism for generating persistent firing in cortical neurons.

The distinct responses of PT and IT neurons to cholinergic modulation suggest that they subserve distinct functions. This is consistent with recent findings showing that, during motor planning and movement, behavior activity with a contralateral population bias arises specifically in PT, but not in IT, neurons (Li et al., 2015). The ability of PT neurons to undergo persistent firing beyond their stimulus input makes them good candidates to contribute to persistent activity that occurs during the delay period in movement planning. Indeed, recent findings show that population activity in PT neurons appears and persists for hundreds of milliseconds before movement onset (Li et al., 2015).

Limitations of the ChAT-ChR2-EYFP mouse line used in our studies

The majority of cholinergic axons in neocortex originate from somata of cholinergic neurons in the basal forebrain, primarily in NB, with a minority originating from cholinergic interneurons and other nuclei within the basal forebrain complex, such as the medial septum (Bigl et al., 1982). ChR2-YFP observed in neocortex in ChAT-ChR2-EYFP mice was expressed mostly by projections from NB and also by local cholinergic interneurons (Zhao et al., 2011). It is therefore impossible to stimulate selectively the projection from NB in these mice, so ACh released from cholinergic interneurons also contributes to the observed responses.

Whereas neither exogenous application of ACh nor optogenetic stimulation of cholinergic fibers matches endogenous ACh release levels (Kolisnyk et al., 2013), both approaches are consistent with a cell-specific effect of cholinergic modulation on L5B corticocollicular and corticocallosal neurons. Moreover, both approaches resulted in consistent findings on the effects of ACh on L5B corticocollicular and corticocallosal neurons. One notable difference between the effects of exogenous application of ACh and optogenetic stimulation of endogenous ACh is the lack of nicotinic responses in a subset of corticocallosal neurons in response to optogenetic stimulation. This difference could be due to desensitization of nAChRs in ChAT-ChR2-EYFP mice due the enhanced cholinergic tone observed in these mice (Kolisnyk et al., 2013).

nAChR- and mAChR-mediated responses in L5 cortical pyramidal neurons

Several studies have used exogenous ACh and one study has used endogenous ACh (Hedrick and Waters, 2015) to study cholinergic neuromodulation in L5 cortical neurons. Exogenous and endogenous ACh causes mAChR-mediated hyperpolarizing and depolarizing responses, as well as facilitation and inhibition in L5 principal neurons of different cortices (Metherate et al., 1992; Gulledge and Stuart, 2005; Gulledge et al., 2007; Hedrick and Waters, 2015). Overall, these responses are consistent with mAChRs mediating slow depolarizing potentials observed in corticocollicular neurons and hyperpolarizing potentials in corticocallosal neurons in L5B AC. However, the biphasic response seen in AC L5 corticocallosal neurons is unique to the AC. In AC L5 corticocallosal neurons biphasic responses consisted of a depolarization followed a hyperpolarization, which were mediated by nAChRs and mAChRs, respectively, whereas in the somatosensory cortex, biphasic responses consisted of a hyperpolarization followed by a depolarization, which were both mediated by mAChRs (Gulledge and Stuart, 2005).

Because we observed the effects of long-lasting depolarization in corticocollicular neurons at different membrane potentials (e.g., long-lasting depolarization at ∼−70 mV and persistent firing near AP threshold in Figs. 7, 8), we suggest that this response is a feature of some corticocollicular neurons and not, for example, a voltage-dependent property of all corticocollicular neurons. In addition, because we never observed this long-lasting mAChR-mediated depolarization in corticocallosal neurons at any membrane potential, this result further supports the notion that this response is specific to only some corticollicular neurons. Although we did not identify any correlations between the variability in the intrinsic properties of corticocollicular with the presence of the long-lasting muscarinic depolarization, our results do not exclude the influence of cell-specific factors mediating this differential response. For example, in layer 2/3 of the AC, a long-lasting depolarization after muscarinic activation is mediated by M1 receptors (Aramakis et al., 1999). Therefore, potential differential expression of muscarinic receptor subtypes within the corticollicular neuronal population could explain the lack of the long-lasting mAChR-mediated depolarization in a subset of corticollicular neurons. However, further studies are needed to elucidate the mAChR subtypes underlying the hyperpolarization and long-lasting depolarization observed in corticocallosal and corticollicular neurons, respectively.

Although we did not investigate the AChR subunit subtypes and the signaling mechanisms mediating the mAChR-evoked potentials, based on studies in other brain regions, we propose that the hyperpolarizing potentials in corticocallosal neurons could be either mediated by activation of either the Gi-coupled M2/M4 receptors or the Gq-coupled M1 receptor that triggers IP3-mediated increase in intracellular Ca2+ and subsequent activation of SK potassium channels (Newberry and Priestley, 1987; Gulledge and Stuart, 2005; Gulledge et al., 2007; Eggermann and Feldmeyer, 2009; Gulledge et al., 2009). In corticollicular neurons, the long-lasting depolarization is potentially mediated by a direct activation of M1 and/or M3 receptors on corticocollicular neurons, which may in turn inhibit KCNQ potassium channels (Delmas and Brown, 2005), or by activation of M1-mediated enhancement of NMDA responses through an IP3-dependent pathway (Aramakis et al., 1999).

Cellular mechanisms underlying system-level effects of ACh in AC

The hypothesized combined effect of mAChRs and nAChRs on AC receptive fields is to reduce receptive field width and to enhance responsiveness within the sharpened receptive field (Metherate, 2011). Several in vivo studies have shown that stimulation of NB enhances, via mAChRs, afferent responses in AC evoked by thalamic (Metherate et al., 1992; Metherate and Ashe, 1993) or acoustic (Edeline et al., 1994; Chen and Yan, 2007) stimulation. This result is consistent with the mAChR-mediated prolonged depolarizing potentials that we observed in corticocollicular neurons. Moreover, sharpening of receptive fields by mAChRs is consistent with the biphasic and inhibitory potentials that we observed in L5B corticocallosal neurons. Although previous studies have shown that nAChRs enhance responsiveness via presynaptic regulation of thalamocortical transmission (Metherate, 2004), our results add an additional mechanism that can enhance responsiveness: the nAChR-mediated depolarizing potentials observed in corticocollicular and L5B corticocallosal neurons also contribute ACh-mediated enhanced responsiveness of AC receptive fields. Together, our results are consistent with in vivo studies revealing sharpening and enhancement of AC receptive fields by ACh and, importantly, provide cellular and synaptic mechanisms via which ACh mediates its effects on AC receptive fields.

Footnotes

This work was supported by National Institutes of Health (Grant DC013272 to T.T.). We thank Dr. Brian Davis for help with anatomical experiments.